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Piperazinyloxoquinoline derivatives, potent and selective inhibitors of HIV-1 transcription
3
4
Molecular Properties of HIV-I Resistant to (+)-Enantinmers and
FROM CARBOVIR TO_ 1592. Robert Vince*, William B. Parker**, and William M. Shannon*" Department of Medicinal Chemistry, College of Pharmacy. University of Minnesota, Minneapolis, MN 55455,* Southern Research Institute. Birmingham, AL.**
Raeemates of Oxathiolane Cytosine Nucleosides and their Fotential for the Treatment of HIV and HBV Infections. Schinazi, R.F., 1~ McMillan, A., 1 Lloyd, RL., jr., 1 Schlueter-Wirtz, %1 Liotta, De., 1 and Chu, C.K." Emory U n i v e r s i t y / V A Medical Center, Decatur, GA, t and U n i v of Georgia, Athens, GA, USA.2
Carbocyclic-2"3' didehydro-2',3'-dideoxyguanosine is the parent compound in a series of 6-substituted-2-amino purine carbocyclic nucleosides (called carbovir, CVR) that are potent and sclecti ve inhibitors of human immunodeflciency virus IVinee etal.. BBRC 156: 10,16, 1988; Vinee and Hua. US patents 4.916.224.4.931.559, 4,950,758, (1990)].
The development of resistant viruses to (-)-Ja-L-oxathiolane cytosine nucleosides produced variants that are highly (> 1,000fold) resistant to 3TC [(-)-BCH-1891 and (-)-FTC, but that remained susceptible other nucleosides, NNRTI, and protease inhibitors, The combination of AZT and 3TC is one of the most successful drag combination for treating HIV infected individuals. especially when combined with protease inhibitors. In cell culture ~he (+)-!3-D-enantiomers and racemates of both FTC and BCH-189 are effective inhibitors of HIV and hepatitis B virus (HBV). However, (+)-BCH-189 was found to be toxic in certain cells such as CEM and human bone marrow cells, whereas (+)-FTC or racemic FTC are essentially non-toxic. In this study, we demonstrate that in HIV 1 infected human PBM cells. (+)-BCH189 readily selected for a virus that was > 8-fold less susceptible to (+)-BCH-189 or racemic BCH-189. DNA sequence analysis of the RT gene amplified from (+)-BCH-189-resistant viruses consistently identified a single mutation at codon 215 from T (ACe) to Y ('I~AC). Clinically this change has been associated with AZTresistance. It was markedly more difficult to select for resistant virus with the 215 mutation with (+)-FTC or racemic BCH-189. although mutations at codon 184 (M to V) were observed with tacemic BCH-189. but not with either (+)-enantiomers Racemic FTC appeared to prevent the development of a mutation at codon 215, but not at 184. These studies demonstrate that it may not always be rational to clinically develop only pure enantiomers of nucleoside antivirals. Unfortunately, one cannot use (+)-BCH-189 or racemic BCH-189 clinically or as part of a combination due to the high toxicity in vitro. However, the potential use of (+)-FTC or raccmic FTC should be considered for the therapy of HIV and HBV (Supported by the Dept. of Veterans Affairs and NIH).
CI NH ©:C
~H
NH{'~N "~,
N
N
NH2 N'~'N~
Wc ha~ e identified the active CVR enantiomer, characterized its ntetabolisnt, identified its active form (CVR-triphosphate) as an inhibitor of reverse transcript~se, and demonstrated its potent s). nerg~ a ith AZT. The 6~alkylamino and 6-alkoxy derivatives ~ ere also active against against HIM The 6-substituted CVR's are metabolized directly to the monophosphates and are subsequently converted to CVR-triphosphate via of CVR monophosphate. The adenosine deaminase inhibitor. EHNA, had no effect on anti-HiV activity of these compounds, while the adenylic acid deaminase inhibitor, 2-deoxycofonnycin. inhibited atttiviral activity. The 6-substituted CVR. 1592, a prodrug of CVR monophosphate, has recently been reported as a clinicall~ active candidate for the treatment of AIDS.
5
6
Identification of the Antiviral Nucleoside Drug Binding Site of HIV-I Integease by Proteolytic Peptide Mapping R Drake. N
Pipcrazin?,lox.quinoline dcri~ ativcs, potent and selective inhibitors nf HIV-I transcription X1 Baba M Okam0lo" M Makino" Y Kmmra T Ikeuchi. 1 Sakagucht and l" Okamolo
Neamati. P. Sunthankar. A Mazumdc-r, Y Ponunier Dep! of Biochemistry. Univ. of,,~kansas for Med Sei. Little Ro~k, AR 722(15 and Laborato~." of Mol Pharm., NCI. NIH. Bethesda, MD. 20892. The recent successes of combination th~apies h.ll'gdt~.tto HIV- I re".'crs¢transcnplase and proleas¢ pro'. tde an intr¢.7.tsedimpetus for idenlt~ing fllV-I integras¢ IttlV-IN~ inhthitors Phosphor~latcxl mctabolitc's of anli~,lfaJ nuck'oside drugs, ~ pifi~.~lby a/idolh) mtdine monophosphate (AZ'f MP). are capable of mhthmng the 3'-ptoc~smg and strand transfer te~:tions of HIV-IN sin a specific Inc, a l l , it3 nucleoh,d¢ site Ioeat~l ~,~,ithin the catal?lie coqe re$tdu~ 51)-212 A pholoafftmb mt,'dogof AZIMP. 5-azido-3'-a/~do-2'.3'..dicl.'oxaurfflme m o n o p h t r ~ e (5N,.,~J.ddUMPI. '.'.as us~J to identt~ this ntlck'ottd¢ s~tc m r~ombinanl WI" and mutant HIV-IN Photointorpo~ation of {~:PISN,AZddb%IPinto ItlV- I inle~'a~. '~,as half-maximal at ~X~p,M. and tt ~,as competitively mhibtt~l by ALl NIP Al.,o. 5N ~AZddUMP v.as an ml~b~torof the 3'-I~Oeessmg and strand transf~ reactions '~,lth 50-70 ~M IC~,~alues N o n - h u e S . high affmtb nlhthtlors thought to bind in the D.D-35-E catal) uc regKm had little iuhthttor). ¢tfi.xt tm photolabehng A n~'~ prot¢ol.~ttc mapping procedure ~as used to identify photocrosslmk~Mpeptides, and by comparison ~ ~th different protease digestions, a Ix'pride region corresponding to ~amno acids 158-170 'o.as det,~n'mir~"das the site of crosshnkmg Structttrall~,.this peptld~ cof[esp~nds tO a surface site Oil the it4 helix dentin adjacent to the catal)tic core D.D-35-E region Site-specific mutants in this region result tn loss of DNA binding and catahtic actr. it_v B~.¢ause most potent HIV-IN mhibitors bind at the catala,t~¢ site and are predominantly Iipophilic. i&'ntifieation of this 158-17(I site veil allo',~ the ft.-sign and testing ofne~x hydrophihc drugs for HIV.IN
"[~¢~wr.i f~t [[u.lo~1 l~etrcnlruse~
('eplter [i~r ('hr~tll~ llra] Disea.~es
5., ,~im t l&,~t,arc h l ~lhnral,~rw~ " [ )mlc hl [~harttlaf ezltic a/( "ll [ td 7~1,~?,I1 ' ;4 gift., a"d Ik,parm,,eln ,~] Lt, de(u/ar (;enem~. \a~zma ('t(v
\~ c bax e found noxcl piperazin) loxoqudloline deri',ati,. es to be polenl uld ~clccHx¢ inlnbitors of ttlV-I replicaliofl in both aculel) and chroni,:;I]1~ Infecled ceils K-12 Ibe inns[ potenl congener of tile series, complctcl) ltdubilcd HIV-I rcplicanon in M O L T 4 cells (aculel) infected ~ith [11 slralll) ;it ~1COllCenlr~llJonsof I] I(i-n 8 uM IAitboulshov.Ingan) C~,loIn\lOll; IIS 5~1'%cffccli\c COllcentranon ( E e l ) and 5n'% c~,totoxic con.~cInrallon ~CC ~ :',ere ~1I)61 and 6 6 uM respectnel'. K-12 ~as also udubnorx lo HI\-I III MT-4 and peripheral blood mononuclear cells It ~i:1~.aCll;Cag;lltlSl ~111HIV-I cllmcal isolate (HE) and H[V-2 (EHO) Tile c01npotlnd colaplelcl3 snpprcsscd lUlnor necrosis faclor ITNF),,,.-induccd IllX-I expression ill latenll} nlfO¢lcd cells IOM-lfl l) and constituhvc \ IIHI ptoduclion In chtollicall~, infccled ceils/MOLT-4/III~d at a concenIrat~on of II g ~tM. as delermined b~, p24 antigen production in culture nllpefll;llalllS Tile HIV-I proleasc inh]bilof VX-47g ~as also effective, hox~c~cr Ihe lal mbibnor Ro24-7-129 shov,¢da;arginal effocl irt OM- I[11 cc/I~ the rcxcr~c Iranscriplasc udlibit0r zido',udlne ~as totalb inacti,.¢ Farti/crnlorc K-I2 could inhibit H1V-I antigen expression in OM-ll)I :t,d MOLI-4/III~ cells, v.hcreas VX-478 did nol affect the anllgen cxi)rc~slOtl;11 lion-to\it concenlratlons Northern blot anal2,sis re;ealed Ihat K 12 ~clccIl~Cl~,prc~cnlcd Ihc accumulatioll of HIV-I mRNA in MOLT4 II[ and FNf-,x-treatcd On-In I celts in a dose-dcpcnaenl fashion [ hc,.c results indicale 1hal ibe plpcrazln~IoxOqulnolineden', alP.cs repro,cut a group of HI\-I inhibuo'fs ;~.ilh :t unique nlechalllsInof action
A42
4
Molecular Properties of HIV-I Resistant to (+)-Enantinmers and
FROM CARBOVIR TO_ 1592. Robert Vince*, William B. Parker**, and William M. Shannon*" Department of Medicinal Chemistry, College of Pharmacy. University of Minnesota, Minneapolis, MN 55455,* Southern Research Institute. Birmingham, AL.**
Raeemates of Oxathiolane Cytosine Nucleosides and their Fotential for the Treatment of HIV and HBV Infections. Schinazi, R.F., 1~ McMillan, A., 1 Lloyd, RL., jr., 1 Schlueter-Wirtz, %1 Liotta, De., 1 and Chu, C.K." Emory U n i v e r s i t y / V A Medical Center, Decatur, GA, t and U n i v of Georgia, Athens, GA, USA.2
Carbocyclic-2"3' didehydro-2',3'-dideoxyguanosine is the parent compound in a series of 6-substituted-2-amino purine carbocyclic nucleosides (called carbovir, CVR) that are potent and sclecti ve inhibitors of human immunodeflciency virus IVinee etal.. BBRC 156: 10,16, 1988; Vinee and Hua. US patents 4.916.224.4.931.559, 4,950,758, (1990)].
The development of resistant viruses to (-)-Ja-L-oxathiolane cytosine nucleosides produced variants that are highly (> 1,000fold) resistant to 3TC [(-)-BCH-1891 and (-)-FTC, but that remained susceptible other nucleosides, NNRTI, and protease inhibitors, The combination of AZT and 3TC is one of the most successful drag combination for treating HIV infected individuals. especially when combined with protease inhibitors. In cell culture ~he (+)-!3-D-enantiomers and racemates of both FTC and BCH-189 are effective inhibitors of HIV and hepatitis B virus (HBV). However, (+)-BCH-189 was found to be toxic in certain cells such as CEM and human bone marrow cells, whereas (+)-FTC or racemic FTC are essentially non-toxic. In this study, we demonstrate that in HIV 1 infected human PBM cells. (+)-BCH189 readily selected for a virus that was > 8-fold less susceptible to (+)-BCH-189 or racemic BCH-189. DNA sequence analysis of the RT gene amplified from (+)-BCH-189-resistant viruses consistently identified a single mutation at codon 215 from T (ACe) to Y ('I~AC). Clinically this change has been associated with AZTresistance. It was markedly more difficult to select for resistant virus with the 215 mutation with (+)-FTC or racemic BCH-189. although mutations at codon 184 (M to V) were observed with tacemic BCH-189. but not with either (+)-enantiomers Racemic FTC appeared to prevent the development of a mutation at codon 215, but not at 184. These studies demonstrate that it may not always be rational to clinically develop only pure enantiomers of nucleoside antivirals. Unfortunately, one cannot use (+)-BCH-189 or racemic BCH-189 clinically or as part of a combination due to the high toxicity in vitro. However, the potential use of (+)-FTC or raccmic FTC should be considered for the therapy of HIV and HBV (Supported by the Dept. of Veterans Affairs and NIH).
CI NH ©:C
~H
NH{'~N "~,
N
N
NH2 N'~'N~
Wc ha~ e identified the active CVR enantiomer, characterized its ntetabolisnt, identified its active form (CVR-triphosphate) as an inhibitor of reverse transcript~se, and demonstrated its potent s). nerg~ a ith AZT. The 6~alkylamino and 6-alkoxy derivatives ~ ere also active against against HIM The 6-substituted CVR's are metabolized directly to the monophosphates and are subsequently converted to CVR-triphosphate via of CVR monophosphate. The adenosine deaminase inhibitor. EHNA, had no effect on anti-HiV activity of these compounds, while the adenylic acid deaminase inhibitor, 2-deoxycofonnycin. inhibited atttiviral activity. The 6-substituted CVR. 1592, a prodrug of CVR monophosphate, has recently been reported as a clinicall~ active candidate for the treatment of AIDS.
5
6
Identification of the Antiviral Nucleoside Drug Binding Site of HIV-I Integease by Proteolytic Peptide Mapping R Drake. N
Pipcrazin?,lox.quinoline dcri~ ativcs, potent and selective inhibitors nf HIV-I transcription X1 Baba M Okam0lo" M Makino" Y Kmmra T Ikeuchi. 1 Sakagucht and l" Okamolo
Neamati. P. Sunthankar. A Mazumdc-r, Y Ponunier Dep! of Biochemistry. Univ. of,,~kansas for Med Sei. Little Ro~k, AR 722(15 and Laborato~." of Mol Pharm., NCI. NIH. Bethesda, MD. 20892. The recent successes of combination th~apies h.ll'gdt~.tto HIV- I re".'crs¢transcnplase and proleas¢ pro'. tde an intr¢.7.tsedimpetus for idenlt~ing fllV-I integras¢ IttlV-IN~ inhthitors Phosphor~latcxl mctabolitc's of anli~,lfaJ nuck'oside drugs, ~ pifi~.~lby a/idolh) mtdine monophosphate (AZ'f MP). are capable of mhthmng the 3'-ptoc~smg and strand transfer te~:tions of HIV-IN sin a specific Inc, a l l , it3 nucleoh,d¢ site Ioeat~l ~,~,ithin the catal?lie coqe re$tdu~ 51)-212 A pholoafftmb mt,'dogof AZIMP. 5-azido-3'-a/~do-2'.3'..dicl.'oxaurfflme m o n o p h t r ~ e (5N,.,~J.ddUMPI. '.'.as us~J to identt~ this ntlck'ottd¢ s~tc m r~ombinanl WI" and mutant HIV-IN Photointorpo~ation of {~:PISN,AZddb%IPinto ItlV- I inle~'a~. '~,as half-maximal at ~X~p,M. and tt ~,as competitively mhibtt~l by ALl NIP Al.,o. 5N ~AZddUMP v.as an ml~b~torof the 3'-I~Oeessmg and strand transf~ reactions '~,lth 50-70 ~M IC~,~alues N o n - h u e S . high affmtb nlhthtlors thought to bind in the D.D-35-E catal) uc regKm had little iuhthttor). ¢tfi.xt tm photolabehng A n~'~ prot¢ol.~ttc mapping procedure ~as used to identify photocrosslmk~Mpeptides, and by comparison ~ ~th different protease digestions, a Ix'pride region corresponding to ~amno acids 158-170 'o.as det,~n'mir~"das the site of crosshnkmg Structttrall~,.this peptld~ cof[esp~nds tO a surface site Oil the it4 helix dentin adjacent to the catal)tic core D.D-35-E region Site-specific mutants in this region result tn loss of DNA binding and catahtic actr. it_v B~.¢ause most potent HIV-IN mhibitors bind at the catala,t~¢ site and are predominantly Iipophilic. i&'ntifieation of this 158-17(I site veil allo',~ the ft.-sign and testing ofne~x hydrophihc drugs for HIV.IN
"[~¢~wr.i f~t [[u.lo~1 l~etrcnlruse~
('eplter [i~r ('hr~tll~ llra] Disea.~es
5., ,~im t l&,~t,arc h l ~lhnral,~rw~ " [ )mlc hl [~harttlaf ezltic a/( "ll [ td 7~1,~?,I1 ' ;4 gift., a"d Ik,parm,,eln ,~] Lt, de(u/ar (;enem~. \a~zma ('t(v
\~ c bax e found noxcl piperazin) loxoqudloline deri',ati,. es to be polenl uld ~clccHx¢ inlnbitors of ttlV-I replicaliofl in both aculel) and chroni,:;I]1~ Infecled ceils K-12 Ibe inns[ potenl congener of tile series, complctcl) ltdubilcd HIV-I rcplicanon in M O L T 4 cells (aculel) infected ~ith [11 slralll) ;it ~1COllCenlr~llJonsof I] I(i-n 8 uM IAitboulshov.Ingan) C~,loIn\lOll; IIS 5~1'%cffccli\c COllcentranon ( E e l ) and 5n'% c~,totoxic con.~cInrallon ~CC ~ :',ere ~1I)61 and 6 6 uM respectnel'. K-12 ~as also udubnorx lo HI\-I III MT-4 and peripheral blood mononuclear cells It ~i:1~.aCll;Cag;lltlSl ~111HIV-I cllmcal isolate (HE) and H[V-2 (EHO) Tile c01npotlnd colaplelcl3 snpprcsscd lUlnor necrosis faclor ITNF),,,.-induccd IllX-I expression ill latenll} nlfO¢lcd cells IOM-lfl l) and constituhvc \ IIHI ptoduclion In chtollicall~, infccled ceils/MOLT-4/III~d at a concenIrat~on of II g ~tM. as delermined b~, p24 antigen production in culture nllpefll;llalllS Tile HIV-I proleasc inh]bilof VX-47g ~as also effective, hox~c~cr Ihe lal mbibnor Ro24-7-129 shov,¢da;arginal effocl irt OM- I[11 cc/I~ the rcxcr~c Iranscriplasc udlibit0r zido',udlne ~as totalb inacti,.¢ Farti/crnlorc K-I2 could inhibit H1V-I antigen expression in OM-ll)I :t,d MOLI-4/III~ cells, v.hcreas VX-478 did nol affect the anllgen cxi)rc~slOtl;11 lion-to\it concenlratlons Northern blot anal2,sis re;ealed Ihat K 12 ~clccIl~Cl~,prc~cnlcd Ihc accumulatioll of HIV-I mRNA in MOLT4 II[ and FNf-,x-treatcd On-In I celts in a dose-dcpcnaenl fashion [ hc,.c results indicale 1hal ibe plpcrazln~IoxOqulnolineden', alP.cs repro,cut a group of HI\-I inhibuo'fs ;~.ilh :t unique nlechalllsInof action
A42